Trypsin inhibitors with insecticidal properties obtained from PENTACLETHRA MACROLOBA

ABSTRACT

Compositions and methods for controlling pests, particularly insect pests, are provided. The compositions comprise proteins isolated from plants of the genus Pentaclethra which exhibit trypsin inhibiting activity. Nucleotide sequences encoding the proteins are also provided. Such sequences find use in transforming organisms for control of pests.

RELATED INVENTION

This application,is a Continuation-In-part of application Ser. No.08/560,727, filed Nov. 20, 1995, now U.S. Pat. No. 5,672,680 andentitled PENTACLETHRA MACROLOBA PROTEIN HAVING INSECTICIDAL PROPERTIES.The specification of the copending application is incorporated herein byreference. This application and the copending application are owned by acommon assignee.

FIELD OF THE INVENTION

The invention relates to methods and materials for controlling insectspecies. In particular, the invention relates to insecticidal proteins,DNA sequences encoding such proteins, and the genetic manipulation ofplants and other organisms.

BACKGROUND OF THE INVENTION

Numerous insect species are serious pests to common agricultural cropssuch as corn, soybeans, peas and similar crops. During the last century,the primary method of controlling such pests has been through theapplication of synthetic chemical insecticidal compounds. However, asthe use of such chemical compounds proliferated and continued, it becameevident that such wide-spread use posed problems with regard to thenon-selectivity of the compounds, the increasing insect resistance tothe chemicals and the environmental affect of such compounds, afterrun-off, on higher order species such as fish and birds among others. Asa result of such problems, other methods of controlling insect pestswere sought and tried.

One such alternative method of pest control has been the use ofbiological organisms which are typically "natural predators" of thespecies sought to be controlled. Such predators may include otherinsects, fungi (milky-spore) and bacteria such as Bacillus thuringiensiscv. Alternatively, large colonies of an insect pest have been captivelyraised, sterilized and released into the environment in the hope thatmating between the sterilized insects and fecund wild insects willdecrease the insect population. While both these approaches have hadsome success, they entail considerable expense and present several majordifficulties. For example, it is difficult both to apply livingbiological organisms to large areas and to cause such organisms toremain in the treated area or on the treated plant species for anextended time. Predator insects can migrate and fungi or bacteria can bewashed off a plant or removed from a treated area by rain. Consequently,while the use of such biological controls has desirable characteristicsand has met with some success, in practice these methods seem severelylimited. However, scientific advances seem to offer new opportunitiesfor controlling insect pests.

Advances in biotechnology in the last two decades have presented newopportunities for pest control through genetic engineering. Inparticular, advances in plant genetics coupled with the identificationof insect growth factors and naturally-occurring plant defensivecompounds or agents offer the opportunity to create transgenic cropplants capable of producing such defensive agents and thereby protectthe plants against insect attack.

Scientists have identified some specific plant components or compoundswhich act as defensive agents to protect a plant from attack by insectpests and pathogens. While such components are usually present at onlylow levels in various plant tissues, some of them are also capable ofbeing induced to higher levels upon attack by an insect pest or apathogen. Examples of such defensive compounds include alkaloids,terpenes, and various proteins such as enzymes, enzyme inhibitors, andlectins.

Of particular interest are enzyme inhibitors that can block enzymaticactivity and inhibit insect growth. For example, trypsin is a digestiveenzyme. Its role in a body is to hydrolyze polypeptides into smallerunits which can then be utilized by the host subject, for example, aninsect. Blocking trypsin activity will inhibit insect growth. A trypsininhibitor (abbreviated TI) is thus a compound which will block ordecrease trypsin protease activity. As a result of such blockage ordecrease in trypsin protease activity, a host subject which has ingestedTI with its food will obtain little or no benefit from the polypeptidescontained in the food. The host may thus fail to grow, mature and mayindeed ultimately starve and die.

Trypsin inhibitors and lectins have been reported in the seeds of anumber of leguminous tropical plants. The proposed role of such trypsininhibitors (TIs) in plant defense has been shown using transgenic plantsexpressing a TI gene. Hilder et al. (1987) Nature 330:160-163,introduced the Bowman-Birk TI gene from soybeans into tobacco plants andshowed that the transgenic plants were able to resist damage from alepidopteran insect. Transformation and expression of other TI genessuch as potato TI I and II also resulted in transgenic plants whichshowed resistance to insect attack. In the same experiments, transgenicplants which contained an unexpressed TI gene were susceptible to insectattack (See, Johnson et al. (1989) Proc. Natl. Acad. Sci. USA86:9871-9875).

Transgenic plants that are resistant to specific insect pests have beenproduced using genes encoding Bacillus thuringiensis (Bt) endotoxins orplant protease inhibitors (PIs). Transgenic plants containing Btendotoxin genes have been shown to be effective for control of someinsects. Effective plant protection using transgenically inserted PIgenetic material has not yet been demonstrated in the field. Whilecultivars expressing Bt genes may presently exhibit resistance to someinsect pests, resistance based on the expression of a single gene mighteventually be lost due to the evolution of Bt resistance in the insects.Thus, the search for additional genes that can be inserted into plantsto provide protection from insect pests are needed.

SUMMARY OF THE INVENTION

Compositions and methods for the control of insects and other pests areprovided. The compositions comprise proteins having pesticidalactivities which can be isolated from leguminous plants. Particularly,the invention identifies and provides trypsin inhibitors obtainable fromthe genus, Pentaclethra, said inhibitors being approximately 38-45 andapproximately 6-9 kilodaltons (kDa) in size. Purified protein, as wellas amino acid and DNA sequence information is provided for proteinshaving insecticidal activity. The DNA sequences encoding the pesticidalproteins can be used to transform plants, bacteria, and other organismsfor the control of pests.

The compositions and methods of the invention may be used in a varietyof systems for controlling plant and non-plant pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an affinity chromatogram of the isolated trypsininhibitors obtained from P. macroloba in accordance with the invention.

FIG. 2A illustrates the SDS-PAGE separation of P. macroloba trypsininhibitors in accordance with the invention.

FIG. 2B illustrates in-gel trypsin inhibitory activity staining.

FIG. 3 illustrates the separation by Sephadex G-50 of two types oftrypsin inhibitors obtained in accordance with the invention.

FIG. 4 illustrates the separation by HPLC of a trypsin inhibitor mixtureobtained according to the invention and heat treated for 3 minutes byboiling in aqueous solution.

FIG. 5 illustrates an inhibition assay of trypsin and P. macrolobatrypsin inhibitors in BAPNA substrate incubated over time.

FIG. 6 provides the amino acid sequence of the PmSTI from P. macroloba(see also SEQ ID NO:2).

FIG. 7 provides the cDNA sequence of the PmSTI from P. macroloba (seealso SEQ ID NO:1).

FIG. 8 provides the amino acid sequence of the N-terminal region of thePmLTI from P. macroloba (see also SEQ ID NO:3).

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for controlling pests, particularly plantpests, are provided. In particular, novel pesticidal proteins areprovided. The proteins are purified from members of the familyLeguminosae, particularly the Leguminous genus Pentaclethra, moreparticularly the species P. macrophylla and P. macroloba.

In accordance with the invention, the pesticidal proteins produced bymembers of the genus Pentaclethra can be isolated by methods known inthe art. In particular, the proteins of the invention have insecticidalactivity. By insecticidal is intended that the proteins of the inventioninhibit insect growth generally leading to death of the insect. Theproteins of the invention exhibit trypsin inhibiting activity. Thus,after ingestion of the proteins by an insect, the insect fails to growand often dies.

Proteins of interest are trypsin inhibitors. Such proteins can be usedfor pest control in plants, particularly by transforming a plant ofinterest with the trypsin inhibitor. Trypsin inhibitors (TIs) fromdifferent plant species have considerably different inhibitive constants(K_(i) values). See, for example, Belitz et al. (1982) Z. Lebensm.Unters.-Forsch 174:442-446 and Christeller et al. (1989) Insect Biochem.19:233-241. The TIs of the invention may be screened to determine thatthe proteins when expressed in a transgenic crop plant will havesufficient resistance to insect attack. Such methods of testing theinsecticidal activity of proteins are available in the art. See, inparticular, the assays described in the Experimental Section.

The insecticidal proteins of the invention may be classified into atleast two classes based upon size of the protein and range of insectskilled upon ingestion of the protein. These two classes include proteinsin the range of about 6 to about 9 kDa designated PmSTI and proteins inthe range of about 38 to about 45 kDa designated PmLTI. Proteins fromsuch classes can be tested for insecticidal activity against a range ofinsects by bioassay techniques known in the art.

The highest concentration of the trypsin inhibitors of the inventionoccurs in plant storage organs such as seeds and tubers. Such tissuesare a source of additional trypsin inhibitors.

"Isolated" means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living animal inits natural state is not "isolated,"but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis "isolated", as the term is employed herein. For example, with respectto polynucleotides, the term isolated means that it is separated fromthe chromosome and cell in which it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

Methods for protein isolation include conventional chromatography,including gel-filtration, ion-exchange, and affinity chromatography, byhigh-performance liquid chromatography, such as reversed-phasehigh-performance liquid chromatography, ion-exchange high-performanceliquid chromatography, size-exclusion high-performance liquidchromatography, high-performance chromatofocusing and hydrophobicinteraction chromatography, etc., by electrophoretic separation, such asone-dimensional gel electrophoresis, two-dimensional gelelectrophoresis, etc. See for example Current Protocols in MolecularBiology, Vols. 1 and 2, Ausubel et al. (eds.), John Wiley & Sons, NY(1988), herein incorporated by reference.

Once purified protein is isolated, the protein, or the polypeptides ofwhich it is comprised, can be characterized and sequenced by standardmethods known in the art. For example, the purified protein, or thepolypeptides of which it is comprised, may be fragmented as withcyanogen bromide, or with proteases such as papain, chymotrypsin,trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem.257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res. 21:209-215).The resulting peptides are separated, preferably by HPLC, or byresolution of gels and electroblotting onto PVDF membranes, andsubjected to amino acid sequencing. To accomplish this task, thepeptides are preferably analyzed by automated sequenators. It isrecognized that N-terminal, C-terminal, or internal amino acid sequencescan be determined. From the amino acid sequence of the purified protein,a nucleotide sequence can be synthesized which can be used as a probe toaid in the isolation of the gene encoding the pesticidal protein.

In the same manner, antibodies raised against partially purified orpurified peptides can be used to determine the spatial and temporaldistribution of the protein of interest. Thus, the tissue where theprotein is most abundant, and possibly more highly expressed can bedetermined and expression libraries constructed. Methods for antibodyproduction are known in the art. See, for example Antibodies, ALaboratory Manual, Harlow and Lane (eds), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988), and the references citedtherein. See also, Radka et al. (1983) J. Immunol. 128:2804; and Radkaet al. (1984) Immunogenetics 19:63. Such antibodies can be used toisolate proteins with similar binding domains and the proteins testedfor activity against insect pests of interest.

It is recognized that any combination of methods may be utilized topurify proteins having pesticidal properties. As an isolation protocolis being determined, the pesticidal activity can be tested for eachfraction of material obtained after each purification step.

Such purification protocols will result in a substantially purifiedprotein fraction. By "substantially purified" or "substantially pure" isintended protein which is substantially free of any compound normallyassociated with the protein in its natural state. "Substantially pure"preparations of protein can be assessed by the absence of otherdetectable protein bands following SDS-PAGE as determined visually or bydensitometry scanning. Alternatively, the absence of otheramino-terminal sequences or N-terminal residues in a purifiedpreparation can indicate the level of purity. Purity can be verified byrechromatography of "pure" preparations showing the absence of otherpeaks by ion exchange, reverse phase or capillary electrophoresis. Theterms "substantially pure" or "substantially purified" are not meant toexclude artificial or synthetic mixtures of the proteins with othercompounds. The terms are also not meant to exclude the presence of minorimpurities which do not interfere with the biological activity of theprotein, and which may be present, for example, due to incompletepurification.

From fragments of the protein, the entire nucleotide sequence encodingthe protein can be determined by PCR experiments. Likewise, fragmentsobtained from PCR experiments can be used to isolate cDNA sequences fromexpression libraries. See, for example, Molecular Cloning, A LaboratoryManual, Second Edition, Vols. 1-3, Sambrook et al. (eds.) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and thereferences cited therein.

In this manner, proteins and the nucleotide sequences encoding suchproteins can be isolated which are inhibitory or toxic to particularinsect species. In particular, proteins and nucleotide sequences whichare inhibitory or toxic to insects of the orders Lepidoptera andColeoptera can be obtained. Such proteins and nucleotide sequences ofthe invention can be utilized to protect plants from pests, includinginsects, fungi, bacteria, nematodes, viruses or viroids, and the like,particularly insect pests. Nematodes include parasitic nematodes such asroot knot, cyst and lesion nematodes.

Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pestsof the invention for the major crops include: Maize: Ostrinia nubilalis,European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea,corn earworm; Spodoptera frugiperda, fall armyworm; Diatraeagrandiosella, southwestern corn borer; Elasmopalpus lignosellus, lessercornstalk borer; Diatraea saccharalis, surgarcane borer; Diabroticavirgifera, western corn rootworm; Diabrotica longicornis barberi,northern corn rootworm; Diabrotica undecimpunctata howardi, southerncorn rootworm; Melanotus spp., wireworms; Cyclocephala borealis,northern masked chafer (white grub); Cyclocephala immaculata, southernmasked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Sovbean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

The nucleotide sequences of the invention can be used to isolate otherhomologous sequences in other plant species, particularly otherLeguminous species, more particularly other Pentaclethra species.Methods are readily available in the art for the hybridization ofnucleic acid sequences. Coding sequences from other plants may beisolated according to well known techniques based on their sequencehomology to the coding sequences set forth herein. In these techniquesall or part of the known coding sequence is used as a probe whichselectively hybridizes to other pesticidal coding sequences present in apopulation of cloned genomic DNA fragments or cDNA fragments (i.e.genomic or cDNA libraries) from a chosen organism.

For example, the entire trypsin inhibitor sequence or portions thereofmay be used as probes capable of specifically hybridizing tocorresponding coding sequences and messenger RNAs. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique and are preferably at least about 10nucleotides in length, and most preferably at least about 20 nucleotidesin length. Such probes may be used to amplify the trypsin inhibitorcoding sequences of interest from a chosen organism by the well-knowprocess of polymerase chain reaction (PCR). This technique may be usedto isolate additional coding sequences from a desired organism or as adiagnostic assay to determine the presence of coding sequences in anorganism.

Such techniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, e.g. Sambrook et al., MolecularCloning, eds., Cold Spring Harbor Laboratory Press (1989)) andamplification by PCR using oligonucleotide primers corresponding tosequence domains conserved among the amino acid sequences (see, e.g.Innis et al., PCR Protocols, a Guide to Methods and Applications, eds.,Academic Press (1990)).

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%Formamide with 5× Denhardt's solution, 0.5 % SDS and 1× SSPE at 37° C.;conditions represented by a wash stringency of 40-45% Formamide with 5×Denhardt's solution, 0.5% SDS, and 1× SSPE at 42° C.; and conditionsrepresented by a wash stringency of 50% Formamide with 5× Denhardt'ssolution, 0.5% SDS and 1× SSPE at 42° C., respectively), to DNA encodingthe insecticidal genes disclosed herein in a standard hybridizationassay. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual 2dEd. (1989) Cold Spring Harbor Laboratory. In general, sequences whichcode for the trypsin inhibitors and other insecticidal proteins of theinvention and hybridize to the gene disclosed herein will be at least50% homologous, 70% homologous, and even 85% homologous or more with thedisclosed sequence. That is, the sequence similarity of sequences mayrange, sharing at least about 50%, about 70%, and even about 85%sequence similarity.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) "referencesequence", (b) "comparison window", (c) "sequence identity", (d)"percentage of sequence identity", and (e) "substantial identity".

(a) As used herein, "reference sequence" is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, "comparison window" means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. NatL. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., USA; the CLUSTAL program is well describedby Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65(1992), and Person, et al., Methods of Molecular Biology 24:307-331(1994); preferred computer alignment methods also include the BLASTP,BLASTN, and BLASTX algorithms. Altschul, et al., J. Mol. Biol.215:403-410 (1990). Alignment is also often performed by inspection andmanual alignment.

(c) As used herein, "sequence identity" or "identity" in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have "sequence similarity" or "similarity". Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

(d) As used herein, "percentage of sequence identity" means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) (i) The term "substantial identity" of polynucleotide sequencesmeans that a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters. Oneof skill will recognize that these values can be appropriately adjustedto determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsequences for these purposes normally means sequence identity of atleast 60%, more preferably at least 70%, 80%, 90%, and most preferablyat least 95%. Polypeptides which are "substantially similar" sharesequences as noted above except that residue positions which are notidentical may differ by conservative amino acid changes.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. to about20° C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent wash conditions are those in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least about 50, 55,or 60° C. However, nucleic acids which do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The terms "substantial identity" in the context of peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

It is recognized that the pesticidal proteins may be oligomeric and willvary in molecular weight, number of promoters, component peptides,activity against particular pests, and in other characteristics.However, by the methods set forth herein, proteins active against avariety of pests may be isolated and characterized.

In one embodiment of the invention, trypsin inhibitors obtainable fromPentaclethra macroloba, are provided. The trypsin inhibitors areapproximately 38-45 and approximately 6-9 kilodaltons (kDa) in size. The38-45 kDa species, called herein PmLTI, comprises a 43 kDa specieshaving a N-terminal sequence ofGlu-Val-Val-Phe-Asp-Phe-Lys-Gly-Asp-Met-Met-Arg-Asn-Gly-Gly-His-Tyr-Tyr-Phe-Phe-Pro-Ala-Ala-Pro-Tyr-Gly-Gly-Gly-Asn-Leu-Leu-Ala-Ala-Ala-Val(shortened nomenclature: EVVFDFKGDMMRNGGHYYFFPAAPYGGGNLLAAAV) SEQ IDNO:3.

The 6-9 kDA species, called herein PmSTI, comprises the following aminoacid sequence set forth in FIG. 6 and SEQ ID NO:2. The cDNA sequence ofPmSTI is provided in FIG. 7 and SEQ ID NO:1.

The proteins of the invention can be used to protect plants againstinsect attack, particularly European corn borer, Helicoverpa zea, andcorn rootworms as well as from nematodes. Such methods are described inmore detail below.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the pesticidal proteins can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example,Kunkel, T. (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al.(1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walkerand Gaastra (eds.) Techniques in Molecular Biology, MacMillan PublishingCompany, NY (1983) and the references cited therein. Thus, the genes andnucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant forms. Likewise, the proteins ofthe invention encompass both naturally occurring proteins as well asvariations and modified forms thereof. Such variants will continue topossess the desired pesticidal activity. Obviously, the mutations thatwill be made in the DNA encoding the variant must not place the sequenceout of reading frame and preferably will not create complementaryregions that could produce secondary mRNA structure. See, EP PatentApplication Publication No. 75,444.

In this manner, the present invention encompasses the pesticidalproteins as well as components and fragments thereof. That is, it isrecognized that component polypeptides or fragments of the proteins maybe produced which retain pesticidal activity. These fragments includetruncated sequences, as well as N-terminal, C-terminal, internal andinternally deleted amino acid sequences of the proteins.

Most deletions, insertions, and substitutions of the protein sequenceare not expected to produce radical changes in the characteristics ofthe protein. However, when it is difficult to predict the exact effectof the substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. That is, the activity can be evaluated byinsect toxicity assay.

The proteins or other component polypeptides described herein may beused alone or in combination with other proteins or agents to controldifferent insect pests. Other insecticidal proteins include those fromBacillus, including δ-endotoxins and vegetative insecticidal proteins,as well as protease inhibitors (both serine and cysteine types),lectins, α-amylase inhibitors, peroxidase, and the like.

In one preferred embodiment, expression of the proteins of the inventionin a transgenic plant is accompanied by the expression of one or moreBacillus thuringiensis (Bt)-endotoxins. This co-expression of more thanone insecticidal principle in the same transgenic plant can be achievedby genetically engineering a plant to contain and express such genes.Alternatively, a plant, Parent 1, can be genetically engineered for theexpression of at least one of the proteins of the invention. A secondplant, Parent 2, can be genetically engineered for the expression ofother principles, such as a Bt-endotoxin. By crossing Parent 1 withParent 2, progeny plants are obtained which express all the genesintroduced into Parents 1 and 2.

The present invention also encompasses nucleotide sequences fromorganisms other than Pentaclethra, where the proteins cross-react withantibodies raised against the proteins of the invention or where thenucleotide sequences are isolatable by hybridization with the nucleotidesequences of the invention. The proteins isolated or those encoded bysuch nucleotide sequences can be tested for pesticidal activity. Theisolated proteins can be assayed for pesticidal activity by the methodsdisclosed herein or others well-known in the art.

Once the nucleotide sequences encoding the pesticidal proteins of theinvention have been isolated, they can be manipulated and used toexpress the protein in a variety of hosts including microorganisms andplants. It is recognized that the proteins and DNA sequences of theinvention may be used alone or in combination with other pesticidalproteins.

The proteins of the invention may be used for protecting agriculturalcrops and products from pests by introduction via a suitable vector intoa microbial host, and said host applied to the environment or plants.Microorganism hosts may be selected which are known to occupy the"phytosphere" (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

The proteins of the invention can be used in expression cassettes forexpression in any host of interest. Such expression cassettes willcomprise a transcriptional initiation region linked to the gene encodingthe pesticidal protein of interest. Such an expression cassette can beprovided with a plurality of restriction sites for insertion of the geneof interest to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes suitable for the particular host organism to be used.

The transcriptional initiation region, or promoter, may be native orendogenous or foreign or heterologous to the host. Additionally, thepromoter may be the natural sequence or alternatively a syntheticsequence. By foreign is intended that the transcriptional initiationregion is not found in the wild-type host into which the transcriptionalinitiation region is introduced. As used herein a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region which is heterologous to the coding sequence. Whileany promoter or promoter element capable of driving expression of acoding sequence can be utilized, of particular interest for expressionin plants are root promoters (Bevan et al. (1993) in Gene Conservationand Exploitation. Proceedings of The 20th Stadler Genetics Symposium,Gustafson et al. (eds.), Plenum Press, New York pp. 109-129; Brears etal. (1991) Plant J. 1:235-244; Lorenz et al. (1993) Plant J. 4:545-554;U.S. Pat. Nos. 5,459,252; 5,608,149; 5,599,670); pith (U.S. Pat. Nos.5,466,785; 5,451,514; 5,391,725); or other tissue specific andconstitutive promoters (See, for example, U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142), herein incorporated by reference.

The transcriptional cassette will include in the 5'-3' direction, theorientation of transcription, a transcriptional initiation region, atranslational initiation region, a DNA sequence of interest, atranslational termination region, and a transcriptional terminationregion functional in plants. The termination region may be native withrespect to the transcriptional initiation region, may be native withrespect to the DNA sequence of interest, or may be derived from othersources. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al., (1991) Mol.Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon etal. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. 1989)Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res.15:9627-9639.

The sequences of the invention can be targeted to the chloroplast. Inthis manner, where the gene of interest is not directly inserted intothe chloroplast, the expression cassette will additionally contain agene encoding a transit peptide to direct the gene of interest to thechloroplasts. Such transit peptides are known in the art. See, forexample, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126;Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al.(1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys.Res Commun. 196: 1414-1421; and, Shah et al. (1986) Science 233:478-481. Plant carotenoid genes useful in the invention may utilizenative or heterologous transit peptides.

The nucleotide sequences encoding the proteins or polypeptides of theinvention are particularly useful in the genetic manipulation of plants.In this manner, the genes of the invention are placed into expressioncassettes for expression in the plant of interest. The cassette willinclude 5' and 3' regulatory sequences operably linked to the gene ofinterest. The cassette may additionally contain at least one additionalgene to be cotransformed into the organism. Alternatively, other gene(s)of interest can be provided on other expression cassettes. Whereappropriate, the gene(s) may be optimized for increased expression inthe transformed plant. That is, the genes can be synthesized using plantpreferred codons for improved expression. Methods are available in theart for synthesizing plant preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known that enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5' leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translational leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. (1989) PNAS USA, 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMVleader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and humanimmunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., andP. Sarnow (1991) Nature, 353:90-94; untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., andGehrke, L., (1987) Nature, 325:622-625; tobacco mosaic virus leader(TMV), (Gallie, D. R. et al. (1989) Molecular Biology of RNA, pages237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S. A.et al. (1991) Virology, 81:382-385). See also, Della-Cioppa et al.(1987) Plant Physiology, 84:965-968. Other methods known to enhanceexpression can also be utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardsthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resection, ligation, PCR, or the likemay be employed, where insertions, deletions or substitutions, e.g.transitions and transversions, may be involved.

The compositions of the present invention can be used to transform anyplant. In this manner, genetically modified plants, plant cells, planttissue, seed, and the like can be obtained. Transformation protocols mayvary depending on the type of plant or plant cell, i.e. monocot ordicot, targeted for transformation. Suitable methods of transformingplant cells include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA, 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al.(1988) Biotechnology, 6:915-921), direct gene transfer (Paszkowski etal. (1984) EMBO J., 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford et al., U.S. Pat. No. 4,945,050; WO91/10725and McCabe et al. (1988) Biotechnology, 6:923-926). Also see, Weissingeret al. (1988) Annual Rev. Genet., 22:421-477; Sanford et al. (1987)Particulate Science and Technology, 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988)Bio/Technology, 6:923-926 (soybean); Datta et al. (1990) Biotechnology,8:736-740(rice); Klein et al (1988) Proc. Natl. Acad. Sci. USA,85:4305-4309(maize); Klein et al. (1988) Biotechnology, 6:559-563(maize); W091/10725 (maize); Klein et al. (1988) Plant Physiol.,91:440-444(maize); Fromm et al. (1990) Biotechnology, 8:833-839; andGordon-Kamm et al. (1990) Plant Cell, 2:603-618 (maize); Hooydaas-VanSlogteren & Hooykaas (1984) Nature (London), 311:763-764; Bytebier etal. (1987) Proc. Natl. Acad. Sci. USA, 84:5345-5349 (Liliaceae); De Wetet al. (1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al., pp. 197-209. Longman, NY (pollen); Kaeppler et al.(1990) Plant Cell Reports, 9:415-418; and Kaeppler et al. (1992) Theor.Appl. Genet., 84:560-566 (whisker-mediated transformation); D=Halluinetal. (1992) Plant Cell, 4:1495-1505 (electroporation); Li et al. (1993)Plant Cell Reports, 12:250-255 and Christou and Ford (1995) Annals ofBotany, 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology,14:745-750 (maize via Agrobacterium tumefaciens); all of which areherein incorporated by reference.

The cells which have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports, 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having the desired phenotypic characteristicidentified. Two or more generations may be grown to ensure that thesubject phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure the desired phenotype or other propertyhas been achieved.

The proteins will be expressed in the transformed organisms in amountsto be toxic to the insects of interest or inhibitory to insect growth.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

P. macroloba seeds were collected at the La Selva Biological Station,Costa Rica and transported to the inventor's laboratories where theywere sliced, lyophilized and stored at -20° C. prior to use. Seedextract was prepared as described by Rathburn et al. (H. Rathburn elal., J. Econo. Ento. (submitted)) with and without heating as notedhere. Heating was accomplished by boiling sliced seeds in 0.1 M Tris-Cl,pH 8.5, 5 mM MgCl₂ before homogenization.

Both trypsin and anhydrotrypsin affinity chromatography, designated TACand ATAC respectively, were used to isolate trypsin inhibitors fromcrude extracts. Anhydrotrypsin was prepared according to the method ofPusztal (A. Pustzai et al., Anal. Biochem. 172: 108-112 (1988)) untilthe trypsin activity was less than 0.5%. Anhydrotrypsin was coupled tocyanogen bromide activated Sepharose 4B according to the manufacturer'sinstructions (Pharmacia). Chromatography columns were equilibrated with0.1 M Tris-Cl, pH 8.5, 5 mM MgCl₂, 3 mM PMFS. Proteins that were boundto the column were eluted with 0.5 M NaOAc buffer containing 0.5 M NaClby decreasing the pH from 7.0 to 2.0. Absorbance at 280 nm (nanometers)was used to follow the elution of the bound proteins. Fractions thatinhibited bovine trypsin were pooled, dialyzed and lyophilized.

Size-exclusion chromatography (SEC) was performed at room temperature,18-28° C., on affinity purified trypsin and anhydrotrypsin fractions. A1.5×100 cm (centimeter) column of Sephadex G-50 was equilibrated with 50mM NaOAc, 0.25 M NaCl, pH 5.0. Two to five milligrams (mg) of affinitypurified trypsin inhibitor was applied to the column and separated at aflow rate of 15 ml/hr. 2.5-ml fractions were collected. Alternatively,trypsin inhibitor protein was separated by HPLC on a Superose 12 column.Samples were heated at 100° C. for 5 minutes in the presence of 2 mM2-mercaptoethanol prior to application to the column. The column wasequilibrated with degassed 50 mM sodium phosphate buffer, pH 7.0, 0.15 MNaCl. The flow rate was controlled at 0.5 ml/min. 0.5 ml fractions werecollected for 20-60 minutes. Fractions which exhibited trypsin activitywere pooled and dialyzed against water.

Ion-exchange chromatography was used to separate large (high molecularweight) trypsin inhibitors from other fractions. Large TIs wereseparated on a 1.5×5 cm anion-exchange column containing BioRad Q5 whichhad been equilibrated with 40 mM ethanolamine-HCl, pH 9.5. TIs wereeluted using a linear gradient from 0 to 0.15 M NaCl. Two-milliliterfractions were collected, tested for inhibition of bovine trypsin andabsorbance at 280 nm was measured.

Crude P. macroloba extracts were extensively dialyzed against deionizedwater at 4EC for several days using a technique combining two molecularweight cutoff (MWCO) membranes or tubings (3,500/10,000 or3,500/14,000). The higher limit MWCO membrane or tubing contained thecrude extract and was placed inside the 3,500 membrane. Deionized waterwas changed at 12 hour intervals. The separated fractions outside thehigher MWCO membrane were collected and subjected to affinitychromatography. When desired, the same tubing combinations can be usedto separate affinity purified trypsin inhibitors. Fractions from SECwere also dialyzed against water using different MWCO membranes.

SDS-PAGE was used to separate components and determine their subunitmolecular weights and purity. The procedure was carried out according tothe method of Laemmli (U.K. Laemmli, Nature 227: 680-685 (1970)) usingtwo types of gel, a 12 to 15% polyacrylamide gel or a 10 to 20% gradientpolyacrylamide gel. Protein samples were denatured by boiling for threeminutes in SDS buffer (BioRad) with 2 mM 2-mercaptoethanol. Selectedsamples, referred to herein as native samples, were not boiled prior toelectrophoresis. The estimated molecular weights of trypsin inhibitorswere estimated by electrophoresis on gradient SDS-PAGE gels of 10-20%polyacrylamide under conditions given by Hammer (B.C. Hammer et al.(1989) Phytochemistry 28: 3019-3026). Proteins were detected by stainingwith Commassie Brilliant Blue R-250 (CBB R-250). Inhibitory activity wasdetected using the assay of Ureil and Berges (J. Ureil et al. (1968)Nature 218: 578-580) as modified by Rathburn (G. Pearce et al. (1993)Plant Physiol. 102: 639-644).

Trypsin inhibitory activity was determined. See, for example Yamamoto etal. (1983) J. Biochem. 94: 849-863; Kim et al. (1985) J. Biochem. 98:435-448; and Richardson et al. (1986) Biochem. Biophys. Acta 872:134-140. The activity of the inhibitors is expressed in Inhibition Units(IU), where 1 IU is the amount of inhibitor needed to reduce BAPNA(e=8.8 μmol⁻¹ cm²) hydrolysis by 1 μmol/min at 25° C.

FIG. 1 illustrates the use of affinity chromatography to isolate trypsininhibitor(s) from P. macroloba. A crude extract was applied directly toa 1×20 cm column and the column was washed until the absorbance of theeluate was of the less than 0.02 at 280 nm. The column was then elutedusing 0.5 M NaOAc, 0.5 M NaCl buffer from pH 2.0 to 7.0 and fractionswere collected.

FIGS. 2A and 2B illustrate SDS-PAGE of P. macroloba inhibitors. In FIG.2A, a silver stain was used. The sample was mixed with non-reducingbuffer (BioRad) and heated for 3 minutes at 100° C. prior toelectrophoresis.

FIG. 2B illustrates in-gel inhibitory activity staining. A sample wasmixed with non-reducing sample buffer and was not heated. The lanesrepresent the following: Lane 1, molecular weight markers; Lane 2, crudePm extract; Lane 3, Affinity-purified PmTI; Lane 4, PmLTI; and Lane 5,PmSTI.

FIG. 1 illustrates the two major peaks that were isolated from trypsinaffinity chromatography (TAC) using pH 3.0 and 2.0 buffers. The pH 2.0fraction was identified as denatured protein and did not inhibit trypsinactivity. The pH 3.0 fraction contains two classes of trypsin inhibitorsdesignated herein as P. macroloba large trypsin inhibitor (PmLTI) and P.macroloba small trypsin inhibitor (PmSTI) as shown in FIGS. 2A and 2B.In addition, several additional polypeptides were obtained. Tests wereconducted to determine whether PmLTI was cleaved by trypsin duringpurification and whether PmSTI was a portion of the degraded PmLTI.Crude extracts were subjected to electrophoresis, and inhibitionactivity was determined using an in-gel trypsin inhibition assay. Twotrypsin inhibitors of the same mobility as that of the affinity purifiedpH 3.0 fraction were detected as shown in FIG. 2. These results indicatethat at least two different trypsin inhibitors, PmLTI and PmSTI, existin P. macroloba seeds.

Anhydrotrypsin affinity chromatography (ATAC) was used to determinewhether PmSTI and the additional polypeptides seen in TAC were theresult of cleavage of PmLTI by fully active trypsin during affinitychromatography. In this purification, the active site of trypsin wasblocked. Trypsin inhibitor isolated by ATAC was considered to be withoutmodification. Five bands were observed on the SDS-PAGE of PmTI isolatedby this procedure and reduced. In contrast, heat denatured Pm TI showedonly one band of Mr=21.5 kDa (data not shown). By comparison to the PmTIisolated from the fully active trypsin affinity column, it was concludedthat Pm TI was degraded by trypsin during affinity chromatographypurification.

PmSTI/PmLTI ratio was found to be very low. In the affinity purifiedproducts, PmSTI could not be detected in stained SDS-PAGE gel despitethe fact that in-gel inhibition assay gave a large clear spot for thisinhibitor.

FIG. 3, gel filtration on Sephadex G-50 carried out at pH 5.0, shows twowell-separated peaks corresponding to PmLTI and PmSTI. SDS-PAGE resultsindicate that peak I is purified PmLTI and peak II is PmSTI, admixedwith a small amount of inactive PmLTI, whose activity was detected bythe in-gel assay of FIG. 2.

FIG. 4 illustrates the separation of heat-treated PmTI by HPLC. Anaqueous sample was boiled for 3 minutes, applied to a Superose column,and eluted.

FIG. 5 is an inhibition assay, over incubation time, of samplescontaining trypsin and PmTI. Samples were prepared using a solution of0.1 M Tris-Cl, pH 9.5. The samples (A, B, C and D) contained 5 μgtrypsin and PmTI in amounts of: (A) 7.2 nanograms, (B) 14.4 nanograms,(C)10.8 nanograms and (D) 7.2 nanograms. BAPNA substrate in the amountof 2.5 mM was added to each sample. Trypsin activity was measured atvarious time points after the addition of the BAPNA substrate.

In order to fully understand the chromatographic behavior of both PmLTIand PmSTI, reduced and native affinity purified trypsin inhibitors wereseparated over a size-exclusion chromatography (SEC) column. Proteinprofiles with and without reduction with p-mercaptoethanol were similar,indicating disulfide bridges are not essential to maintain the dimericstructures. The heat stability of PmSTI, discussed below, can be used todistinguish PmSTI from PmLTI and other trypsin inhibitors. A sample ofPmSTI was heat treated, chromatographed on a SEC column and showed threeproduct peaks. Only peak II was active, indicating that this peakcorresponds to PmSTI. Peak III was close in molecular weight to Peak II,but did not exhibit any inhibition activity.

PmSTI is a small polypeptide with a subunit in the 6-9 kDa range,generally about 7 kDa molecular weight, and is present in lowconcentrations in P. macroloba seeds. Large quantities of PmSTI can beobtained by first dialyzing crude extracts using a double dialysismethod. PmSTI, along with other low molecular weight proteins, dialyzedout of the 12-14 kDa membrane and was collected within a 3.500 MWCOmembrane. The PmSTI fraction was further purified by affinitychromatography followed by gel filtration.

The subunit molecular weight of the trypsin inhibitors was determined by10-20% SDS-PAGE. The two iso-inhibitors (isoforms) constituting PmLTIwere found to have molecular weights of approximately 43 and 39 kDa.Denaturing the PmLTI fraction by heating converted it into a specieshaving a molecular weight of about 21.5 kDa. In-gel trypsin inhibitionassay of PmLTI separated on an IEF gel indicates that it is composed oftwo active proteins of pI 8.8 and 8.5. Similar testing of PmSTIindicates it is a low molecular weight polypeptide having a molecularweight of about 7 kDa and contains two active proteins of pI 8.2 and5.0. [Data not shown]. These results indicate both PmLTI and PmSTIcontain two iso-inhibitors of nearly the same molecular weight.

Heating affinity purified P. macroloba TI's has a profound effect ontheir inhibition activity. A crude extract, containing both PmLTI andPmSTI, had an inhibition activity of 2.2 IU/mg TI before heating and 3.7IU/mg TI after heating at 70° C. for 5 minutes. PmLTI completely losesactivity after heating at 100° C. for 5 seconds. In contrast, PmSTIretained activity after boiling at 100° C. for 30 minutes. PmSTI didlose activity after boiling for 5 minutes under reducing conditions.

Amino Acid Sequence Information

The N-terminal sequence of PmLTI has been determined and is shown, inFIG. 8 and SEQ ID NO:3. The Edman degradation method was used for aminoacid sequencing. The sequencing was performed on an Applied Biosystems477A Protein Sequencer having a 120A Analyzer.

Internal Sequences

Internal amino acid sequence data was determined after cleavage of theprotein with cyanogen bromide and separation of the fragments by SDSPAGE. peptides were blotted to PVDF and subjected to automated sequenceanalysis.

Enzymatic Assay of the Inhibitory Activity of Pm TIs

The inhibitory activity of P. macroloba TIs was determined by enzymaticassay using the method and materials described by J. Chun et al. (1994)J. Econo. Ento. 87: 1754-1760, which include the procedures of: C. J.Lenz et al. (1979) J. Insect Physiol. 25: 487-494; Y. Takesue et al.(1989) J. Biochem. 105: 998-1001; S. E. McEwen et al. (1980) InsectBiochem. 10: 563-567; J. F. Myrtle and W. J. Zell (1980) Clin. Chem. 21:1469-1473; and K. K. Thomas and J. L. Nation (1984) Comp. Biochem.Physiol. A Comp. Physiol. 79:297-304. For these enzyme inhibitionstudies, purified TI's were incubated with appropriate amounts of bufferand bovine trypsin or Helicoverpa zea (H. zea) midgut trypsin at 27° C.The substrates were then added and the reactions were performed asdescribed in the referenced method(s).

The results of inhibitory activity assays of the P. macroloba TI'sagainst bovine and H. zea trypsins are given in Table 1. The specificactivity values were 2.78 IU/mg TI for bovine trypsin and 3.93 IU/mg TIfor H. zea midgut trypsin. The specific activity of PmSTI was 50.94IU/mg TI for bovine trypsin and 14.23 IU/mg TI for H. zea. PmTIsobtained from the fully active trypsin affinity column (TAC) show lowertrypsin inhibition activity than PmTIs obtained from the anhydrotrypsinaffinity column (ATAC). This suggests that PmTI is modified by trypsinin such a manner as to reduce the inhibitory effectiveness of the PmTI.It was also found that PmTI was completely bound to trypsin in about 10minutes.

                  TABLE 1                                                         ______________________________________                                        Inhibitory Activity of PmTIs and Soybean TI                                                 Bovine Tr..sup.1                                                                             H.zea Midgut Tr..sup.1                           Sample Protein (mg)                                                                             1A.sup.2 SA.sup.3                                                                             1A.sup.2                                                                            SA.sup.3                              ______________________________________                                        A.sup.4                                                                              3.1        0.119    0.038  0.245 0.079                                   B.sup.5 2.0 4.959 2.48 7.457 3.729                                            C.sup.6 2.0 5.567 2.784 7.855 3.928                                           D.sup.7 0.6 30.566 50.940 8.538 14.230                                        E.sup.8 N/A N/A 1.016 N/A 0.845                                               F.sup.9 N/A N/A 5.030 N/A 2.101                                             ______________________________________                                         Notes:                                                                        .sup.1 Tr = trypsin.                                                          .sup.2 IA = Inhibitory Activity in IUs.                                       .sup.3 SA = Specific Activity in IU/mg protein.                               .sup.4 A = Crude Extract.?                                                    .sup.5 B = Affinity purified TI.                                              .sup.6 C = PmLTIs.                                                            .sup.7 D = PmSTIs.                                                            .sup.8 E = Kunitz TI.                                                         .sup.9 F = BowmanBirk TI.                                                

Biological Assays with PmTIs

Neonate insect pests were reared on artificial diets containing PmTIs,either crude or purified as taught herein, which were either topicallyapplied to the diet surface or incorporated into the diet as taught byCzapla and Lang (T. H. Czapla and B. A. Lang, J. Econo. Ento. 83 (6):2480-2485 (1990)). The culture tray used in the bioassays was dividedinto treatment groups. One or a plurality of PmTIs were screened in eachtray, each PmTI being applied to a plurality of cells. Each cell wasinfested with one neonate larvae. A Mylar film with ventilation holeswas affixed to the top of each tray to prevent escape.

For the topical assays, the TI was prepared in 0.1 M phosphate bufferedsaline (PBS) buffer (pH 7.8) as a 2% solution. Seventy-five microlitersof solution were pipetted onto Stoneville medium in each cell. Theculture tray was rotated to ensure equal distribution of the inhibitor.The cells were infected and sealed as described above. The control was75 μl of 0.1 M PBS per cell.

For the diet incorporation assays, Stoneville medium was prepared instandard fashion but with only 90% of the prescribed water. PmTI wasadded such that the amount in the diet was in the range of 1-5 μg/g. Thecontrol treatment consisted of 0.9 ml PBS buffer added to 8.1 g ofmedium. The medium was poured into the cells and the cells were theninfested and covered as described above. Insect weights were determinedat Day 7 and are given in Tables 2-4. The data in Tables 2 and 3 wereobtained using extracts from P. macroloba which contain both the PmLTIand the PmSTI component. The data in Table 4 was obtained using PmTIthat was separated into its PmLTI and PmSTI components.

The data in Table 4 were obtained using corn rootworm (CRW) neonatelarvae. While the data in Table 4 indicates that PmLTI is effectiveagainst CRW and PmSTI is not effective against CRW, the data in Table 8indicates that PmSTI is effective against other insect pests.

                  TABLE 2                                                         ______________________________________                                        European Corn Borer Bioassay with PmTIs                                           μg TI/cup                                                                            Avg. Wt.   % Wt. Change                                                                           % Mortality                                 ______________________________________                                        A. Topical application - Weights at Day 7                                          0        4.93 mg    --       6.25                                          10 4.55 mg -7.7 6.25                                                          25 3.99 mg -19.1 6.25                                                         50 3.86 mg -21.7 6.25                                                       B. Incorporated into diet - Weights at Day 7                                    Assay 1                                                                              0        5.07 mg  --       6.25                                         50 6.45 mg +27.2 18.75                                                       100 3.35 mg -33.9 6.25                                                        250 4.l8 mg -17.6 25.00                                                     Assay 2                                                                            0        4.88 mg    --       25.00                                         100 3.41 mg -30.1 18.75                                                       250 2.97 mg -39.1 6.25                                                        500 2.71 mg -44.5 43.75                                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        H.zea Bioassay with PmTIs                                                       Incorporated into Diet - Weight at Day 7                                      μg/ml         Avg. Wt. % Wt. Change                                      ______________________________________                                        Assay 1                                                                         0                92 mg    --                                                  1.5 101 mg +9.9                                                               3.0 101 mg +9.8                                                               6.0 54 mg -41.3*                                                            Assay 2                                                                         0                56 mg    --                                                  3 55 mg -1.8                                                                  6 56 mg 0                                                                     9 36 mg -35.7                                                               Assay 3                                                                         0                182 mg   --                                                  10 198 mg +8.8                                                                45 243 mg +33.5                                                             ______________________________________                                         Note:                                                                         *Comparison significant at the 0.05 level with Sheffe=s test.            

                  TABLE 4                                                         ______________________________________                                        Corn Rootworm Bioassay with PmLTI and PmSTI*                                    PmTI, mg/ml Weight   % Loss v. Control                                                                         % Mortality                                ______________________________________                                        Control, O                                                                              2.8      --            7                                              LTI, 0.2 2.8 0 29                                                             LTI, 0.3 2.8 0 29                                                             LTI, 0.8 0.7 83 57                                                            Control, 0 3.2 -- 0                                                           STI, 0.6 3.2 0 0                                                            ______________________________________                                         Note:                                                                         *Assay conducted with corn rootworm (CRW) neonate larvae. The PmLTI and       PmSTI were incorporated into the diet as described above.                

Trypsin Inhibitor Comparisons

Tables 5, 6 and 7 compare PmTI to Bowman-Birk and Kunitz trypsininhibitors. The results for bovine chymotrypsin in Table 5 indicate thatthe activity of PmTI falls between that of Bowman-Birk and Kunitz, theBowman-Birk being the least active. The results for the inhibition of H.zea midgut trypsin in Table 6 indicate that the activity of PmTI at pH8.0 lies between that of the Bowman-Birk and Kunitz inhibitors, and atpH 9.5 and 10.0 PmTI has a higher level of activity. The results for theinhibition of bovine trypsin in Table 7 indicate that the activity ofPmTI falls between that of the Bowman-Birk and Kunitz T is.

                  TABLE 5                                                         ______________________________________                                        Micrograms of Trypsin Inhibitors to Inhibit                                     1.6 μg Bovine Chymotrypsin by 50%                                                P. macroloba TI                                                                            Bowman-Birk TI                                                                              Kunitz TI                                  ______________________________________                                        pH 8.0* 8.6          1.3           11.8                                         pH 9.0* 4.4 1.0 8.4                                                           pH 9.5* 2.1 1.0 23.6                                                        ______________________________________                                         Note:                                                                         *A11 solutions are 0.1 M TrisC1.                                         

                  TABLE 6                                                         ______________________________________                                         Nanogramsof Trypsin Inhibitors to Inhibit                                      H.zea Midgut Trypsin by 50%                                                         P. macroloba TI                                                                            Bowman-Birk TI                                                                              Kunitz TI                                  ______________________________________                                        pH 8.0* 660          744           323                                          pH 9.5* 939 974 1467                                                          pH 10.0* 925 1196 1401                                                      ______________________________________                                         Note:                                                                         *A11 solutions are 0.1 M TrisC1.                                         

                  TABLE 7                                                         ______________________________________                                        Nanograms of Trypsin Inhibitors to Inhibit                                      5 μg Bovine Trypsin by 50%                                                       P. macroloba TI                                                                            Bowman-Birk TI                                                                              Kunitz TI                                  ______________________________________                                        pH 8.0* 835          754           1551                                         pH 9.5* 765 637 1071                                                          pH 10.0* 787 754 1264                                                       ______________________________________                                         Note:                                                                         *A11 solutions are 0.1 M TrisC1.                                         

Table 8 B presents bioassay data for the Pm small trypsin inhibitor(PmSTI) against both European corn borer (Ostrina nubilalis) and thebacterial feeding nematode Caenorhabditis elegans (C. elegans). C.elegans is used by those in the art as a model system to identifyfeeding factors to control plant parasitic nematodes such as root knot,cyst and lesion nematodes.

The data in Table 8 A shows that the PmSTI gene expressed in E. coli asa fusion protein reduces the growth and survival of Ostrina nubilaliswhich have been reared on diets containing heat-killed E. coli cellsexpressing the protein. All cells were induced with IPTG. The solublePmSTI content of the cells was very low. The methods for growing the E.coli cells are well known to those skilled in the art. Typically, theweight of larvae fed E. coli cells containing the PmSTI gene was lessthan half the weight of larvae fed cells which did not contain the PmSTIgene.

As a result of the low concentration of soluble PmSTI in the E. colicells, the differences in weight between larvae fed cells containingPmSTI and the control larvae are of more significance than the mortalityrate. The data clearly indicates that ingestion of PmSTI, even at lowconcentrations, has a significant effect on larval growth. The inabilityof larvae to gain weight and mature will ultimately affect theirsurvival and ability to reproduce. This observation is in accordancewith the data presented in Tables 2 and 4.

                  TABLE 8                                                         ______________________________________                                        PmSTI Bioassay Data                                                           ______________________________________                                        A. European corn borer                                                                             % Mortality                                                                             Larval Wt..sup.1                               ______________________________________                                          Control,.sup.2 no PmSTI gene 25 7.6                                           Buffer control, no gene 13.3 7.9                                              PmSTI gene.sup.3 18.7 3.5                                                     PmSTI plus kanamycin 18.7 3.5                                               ______________________________________                                        B. C. elegans                                                                                      % Mortality.sup.4                                        ______________________________________                                          Control --                                                                    PmSTI gene 98.8                                                             ______________________________________                                         Notes:                                                                        .sup.1 Larval weight at the end of the 7 day test.                            .sup.2 Heatkilled E. coli cells without PmSTI gene.                           .sup.3 Heatkilled E. coli cells expressing PmSTI gene.                        .sup.4 Mortality at end of 10 day test.                                  

The PmSTI protein was cloned and the amino acid sequence was deducedfrom the sequence of the cDNA clone as shown in FIG. 6 and SEQ ID) NO:2. The cDNA sequence is given in FIG. 7 and SEQ ID NO: 1.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 558 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: PENTACLETHRA - # MACROLOBA                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 25..387                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GGCACGAGGA GAGAGAGACA GAAG ATG GGT TTG AAG AAG G - #CG ACC ATG GTG            51                                                                                        - #         Met Gly Leu Lys Lys - #Ala Thr Met Val                            - #           1       - #        5                           - - AAG GTA GGT GTA GTG CTG TTC CTG ATG GCC CT - #C ACT GCA ACT GTG GAG           99                                                                       Lys Val Gly Val Val Leu Phe Leu Met Ala Le - #u Thr Ala Thr Val Glu            10                 - # 15                 - # 20                 - # 25       - - GGC CGC TTC GAT TCG AAC ACG TTA CTT GCT CA - #G GTG ATG ATG AAG GAG          147                                                                       Gly Arg Phe Asp Ser Asn Thr Leu Leu Ala Gl - #n Val Met Met Lys Glu                            30 - #                 35 - #                 40              - - AAT GGT GAA CCC AAC TAC TTC ATC AAG TCC AC - #C ACC ACC GCC TGC TGC          195                                                                       Asn Gly Glu Pro Asn Tyr Phe Ile Lys Ser Th - #r Thr Thr Ala Cys Cys                        45     - #             50     - #             55                  - - GAC AAC TGC CCT TGC ACA AAG TCA AAC CCA CC - #T CAA TGC CAA TGC AAT          243                                                                       Asp Asn Cys Pro Cys Thr Lys Ser Asn Pro Pr - #o Gln Cys Gln Cys Asn                    60         - #         65         - #         70                      - - GAT TGG AAA GAA ACT TGC CAC TCC GCT TGT AA - #G ACC TGT ATT TGC AGG          291                                                                       Asp Trp Lys Glu Thr Cys His Ser Ala Cys Ly - #s Thr Cys Ile Cys Arg                75             - #     80             - #     85                          - - GCA ATA TAT CCT CCA CAG TGT CGT TGT TTT GA - #T ACC AAC AAC TTC TGC          339                                                                       Ala Ile Tyr Pro Pro Gln Cys Arg Cys Phe As - #p Thr Asn Asn Phe Cys            90                 - # 95                 - #100                 - #105       - - TAT CCT CCT TGC CCC TCT TCT GCT GCC AAA CC - #T CAA CTT GCG AAC TGA          387                                                                       Tyr Pro Pro Cys Pro Ser Ser Ala Ala Lys Pr - #o Gln Leu Ala Asn  *                            110  - #               115  - #               120              - - TCGTCGTTAA TGGTGTGATG TTATGTGAAC GAAGCCCTCT ACTGCTCTAG GC -             #TTTGTTTC    447                                                                 - - TATATATGTA CGTGAATGTG AAGCATATCT AATAAAATAA GATATCGTGG GC -            #CTTTCTTC    507                                                                 - - CAGTTTGCTT TTTGCAAACT GGCTGCTTGC AGGCTCTTGA TCTTCTTCAA A - #                558                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  120 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Gly Leu Lys Lys Ala Thr Met Val Lys Va - #l Gly Val Val Leu Phe        1               5 - #                 10 - #                 15              - - Leu Met Ala Leu Thr Ala Thr Val Glu Gly Ar - #g Phe Asp Ser Asn Thr                   20     - #             25     - #             30                  - - Leu Leu Ala Gln Val Met Met Lys Glu Asn Gl - #y Glu Pro Asn Tyr Phe               35         - #         40         - #         45                      - - Ile Lys Ser Thr Thr Thr Ala Cys Cys Asp As - #n Cys Pro Cys Thr Lys           50             - #     55             - #     60                          - - Ser Asn Pro Pro Gln Cys Gln Cys Asn Asp Tr - #p Lys Glu Thr Cys His       65                 - # 70                 - # 75                 - # 80       - - Ser Ala Cys Lys Thr Cys Ile Cys Arg Ala Il - #e Tyr Pro Pro Gln Cys                       85 - #                 90 - #                 95              - - Arg Cys Phe Asp Thr Asn Asn Phe Cys Tyr Pr - #o Pro Cys Pro Ser Ser                  100      - #           105      - #           110                  - - Ala Ala Lys Pro Gln Leu Ala Asn                                                  115          - #       120                                             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pentaclethra - # macroloba                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Glu Val Val Phe Asp Phe Lys Gly Asp Met Me - #t Arg Asn Gly Gly His      1               5   - #                10  - #                15               - - Tyr Tyr Phe Phe Pro Ala Ala Pro Tyr Gly Gl - #y Gly Asn Leu Leu Ala                  20      - #            25      - #            30                   - - Ala Ala Val                                                                      35                                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated nucleotide sequence encoding aprotein having insecticidal and trypsin inhibiting activity, whereinsaid protein has an amino acid sequence comprising the sequence setforth in SEQ ID NO:
 2. 2. A vector comprising the nucleotide sequence ofclaim
 1. 3. A vector comprising a nucleotide sequence which encodes apolypeptide having insecticidal and trypsin inhibiting activity andwhich hybridizes to the nucleotide sequence of claim 1 under stringentconditions defined by a wash stringency of 0.3 M NaCl, 0.03 M sodiumcitrate, 0.1% SDS at 70° C.
 4. A stably transformed plant comprising inits genome a chimeric gene comprising a nucleotide sequence encoding aprotein having insecticidal activity wherein said protein is about 38-45kDa in size and exhibits trypsin inhibiting activity, wherein saidnucleotide sequence encodes the contiguous amino acids set forth in SEQID NO:
 3. 5. Seed of the plant of claim
 4. 6. The plant of claim 4wherein said plant is a dicot.
 7. The plant of claim 4 wherein saidplant is a monocot.
 8. The plant of claim 7 wherein said monocot ismaize.
 9. Seed of the plant of claim
 8. 10. An isolated nucleotidesequence encoding a protein having insecticidal and trypsin inhibitingactivity said sequence comprising the DNA sequence set forth in SEQ IDNO:
 1. 11. The nucleotide sequence of claim 1, wherein said sequencecomprises the DNA sequence set forth in SEQ ID NO:
 1. 12. An isolatednucleotide sequence which encodes a polypeptide having insecticidal andtrypsin inhibiting activity and which hybridizes to the nucleotidesequence of claim 1 under stringent conditions defined by a washstringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 70° C. 13.The vector of claim 2, wherein said nucleotide sequence comprises theDNA sequence set forth in SEQ ID NO:
 1. 14. A stably transformed plantcomprising in its genome a chimeric gene, said chimeric gene comprisingthe nucleotide sequence of claim
 1. 15. Seed of the transformed plant ofclaim
 14. 16. The transformed plant of claim 14, wherein said nucleotidesequence is the sequence set forth in SEQ ID NO:
 1. 17. Seed of thetransformed plant of claim
 16. 18. The transformed plant of claim 16wherein said transformed plant is a dicot.
 19. The transformed plant ofclaim 16 wherein said transformed plant is a monocot.
 20. Thetransformed plant of claim 19 wherein said monocot is maize.
 21. Seed ofthe transformed plant of claim 20.